Purification and Properties of a D(-)-β-Hydroxybutyric Dimer Hydrolase from Rhodospirillum rubrum<sup>*</sup>

Merrick, Jason R. W.; Yu, Chi Ing

doi:10.1021/bi00875a026

Cited by 38 publications

(19 citation statements)

References 12 publications

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“…Although the physiological role of the 3HB-oligomer hydrolase is not clear, it may be important to study this hydrolase in relation to extracellular degradation of PHB. Similar enzymes were found within the bacterial cells (8,16,25). Pseudomonas sp.…”

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confidence: 71%

Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene

1997

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An extracellular D-(؊)-3-hydroxybutyrate oligomer hydrolase was purified from a poly(3-hydroxybutyrate)-degrading bacterium, Pseudomonas sp. strain A1. The purified enzyme hydrolyzed the D-(؊)-3-hydroxybutyrate dimer and trimer at similar rates. The enzyme activity was inhibited by a low concentration of diisopropylfluorophosphate. The molecular weight of the hydrolase was estimated to be about 70,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 10-kbp DNA fragment of A1 was detected by hybridization with the gene (2 kbp) of an extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. Subsequent subcloning showed that a SmaI-KpnI fragment (2.8 kbp) was responsible for expression of the hydrolase in Escherichia coli and an in vitro transcription-translation system. The expressed protein detected by immunostaining had the same molecular weight as the purified enzyme from A1. The protein band detected in the in vitro transcription-translation system had a molecular size of 72 kDa. The nucleotide sequence of the SmaI-KpnI fragment was determined, and one open reading frame (2,112 nucleotides) was found. It specifies a protein with a deduced molecular weight of 72,876 (704 amino acids). In this sequence, the consensus sequence of serine-dependent hydrolysis, G-X-S-X-G, did not exist.Poly-3-hydroxybutyrate (PHB) is a unique intracellular reserve of organic carbon and/or chemical energy found in a wide variety of bacteria (6, 9) and is regarded as a potential biodegradable thermoplastic not derived from petroleum (12). Recently similar bacterial polyesters have been found, and these polyesters including PHB were called "polyhydroxyalkanoates" (PHAs) (7). Extracellular PHB metabolism is important in the application of these bacterial polyesters as biodegradable plastics. Many microorganisms that are capable of degrading PHB exist in the environment, and they secrete PHB depolymerases to degrade this polymer. There have been many studies on the extracellular PHB depolymerases from several bacteria and fungi (2,3,4,11,15,22,26,29). Genes for some enzymes were also cloned and sequenced (10,19,21). Alcaligenes faecalis T1, a PHB-degrading bacterium, secretes a D-(Ϫ)-3-hydroxybutyric acid oligomer hydrolase (3HB-oligomer hydrolase) (EC 3. 1.1.22) (23) besides a PHB depolymerase. Although the physiological role of the 3HB-oligomer hydrolase is not clear, it may be important to study this hydrolase in relation to extracellular degradation of PHB. Similar enzymes were found within the bacterial cells (8,16,25). Pseudomonas sp. strain A1 was isolated from activated sludge as a PHB-degrading bacterium (22) and was found to have an extracellular 3HB-oligomer hydrolase besides a PHB depolymerase. In this communication, we describe the purification and characterization of the 3HB-oligomer hydrolase and cloning and sequencing of its gene. MATERIALS AND METHODSBacterial strains, plasmids, and culture. Pseudomonas sp. strain A1 was isolated from activated sludge of the sewage treatment facility of...

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confidence: 71%

Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene

1997

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“…The enzyme from Z. ramigera resembled the dimer hydrolase from Rlzodospirillum rubrum [7] in pH optimum (8.0 versus 8.4), K, for DD-dimer (0.59 mM versus 0.25 mM) (Table 3), susceptibility to diisopropylfluorophosphate, and resistance to sulfhydryl reagents.…”

Section: Discussionmentioning

confidence: 93%

Purification and Properties of d(−)‐3‐Hydroxybutyrate‐Dimer Hydrolase from Zoogloea ramigera I‐16‐M

Tanaka

Saito

Fukui

et al. 1981

European Journal of Biochemistry

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u( -)-3-Hydroxybutyrate-dimer hydrolase from Zoogloea ramigera I-16-M was purified 7000-fold to electrophoretic homogeneity. The molecular weight of the purified enzyme was 28000 as determined by Sephadex G-100 gel filtration, and 30 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric point was at pH 5.7. The pH optimum for the enzyme reaction was 8.0. The dimer hydrolase was stereospecific forIn addition, the purified hydrolase hydrolyzed several oligomeric esters Of D( -)-3-hydroxybutyric acid (DDD-trimer, DmD-tetramer and DDDDD-pentamer) faster than DD-dimer. Time course experiments with these oligomers and analysis of hydrolytic products of DmD-tetramer methyl ester with the hydrolase indicated that the enzyme attacked these substrates from the free hydroxyl terminus, releasing monomer units one at a time.The polymeric ester of D( -)-3-hydroxybutyric acid, poly(3-hydroxybutyrate), is accumulated by diverse species of bacteria as a principal intracellular reserve of organic carbon, and is digested when the external carbon supply becomes limited [I -31. However, owing to the extreme complexity of the initial stage of degradation, the enzymatic mechanism of poly(3-hydroxybutyrate) digestion in bacterial cells has not been fully clarified yet demonstrated that poly(3-hydroxybutyrate) granules isolated from Bacillus megaterium KM, which did not undergo self-digestion, were hydrolyzed by a depolymerizing enzyme system present in the soluble fraction of poly( 3-hydroxybutyrate)depleted Rhodospirillum rubrum cells, yielding D( -)-3-hydroxybutyrate as a main product and its dimeric ester (DD-dimer) as a minor product [4]. The latter product was further hydrolyzed to the monomer by a hydrolase (hydroxybutyrate-dimer hydrolase) partially purified from R. rubrum. However, this enzyme hydrolyzed the presumed trimeric ester of D( -)-3-hydroxybutyrate (DDD-trimer) faster than DD-dimer [7]. In contrast, a similar dimer hydrolase partially purified from Pseudomonas lemoignei did not attack DD-trimer to an appreciable extent [S].During studies on the pathway of poly(3-hydroxybutyrate) synthesis in Zoogloea ramigera I-16-M isolated from activated

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“…However, it was very often observed that the intracellular level of PHB was significantly decreased when the carbon source of the meCorrespondence to: J. M. Lebeault dium was exhausted and/or nitrogen source was present in excess during the culture of PHB-producing microorganisms (Schlegel et al 1961;Dawes and Senior 1973;Senior and Dawes 1973). This phenomenon probably resuited from the action of enzymes involved in the degradation pathway of PHB, such as depolymerase and D(-)-fl-hydroxybutyric acid (Dfl-HB) dehydrogenase (fl-HBD, Merrick and Doudoroff 1964;Merrick and Yu 1966;Tanaka et al 1981;Delafield et al 1965;Nakada et al 1981).…”

Section: Introductionmentioning

confidence: 99%

Partial purification and characterization of ?-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium, Pseudomonas 135

Daniel

Barbotin

Kim

et al. 1992

Appl Microbiol Biotechnol

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An intracellular enzyme, D(-)-fl-hydroxybutyric acid dehydrogenase involved in an intracellular poly-D(-)-fl-hydroxybutyric acid degradation was isolated from a facultative methylotrophic bacterium, P s e u d o m o n a s 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to l l.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of D(-)-fl-hydroxybutyric acid (Dfl-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5-6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at -20 ° C. The Km values for oxidation and reduction reactions were determined as 1.84 mM for Dfl-HB, 0.244 mM for NAD +, 0.319 mM for acetoacetate and 0.032 mM for NADH, respectively. It was also found that D-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mM for D-lactate, 0.196mM for NADH and 1.82mM for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive.

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Purification and Properties of a D(-)-β-Hydroxybutyric Dimer Hydrolase from Rhodospirillum rubrum^*

Cited by 38 publications

References 12 publications

Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene

Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene

Purification and Properties of d(−)‐3‐Hydroxybutyrate‐Dimer Hydrolase from Zoogloea ramigera I‐16‐M

Partial purification and characterization of ?-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium, Pseudomonas 135

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Purification and Properties of a D(-)-β-Hydroxybutyric Dimer Hydrolase from Rhodospirillum rubrum*

Cited by 38 publications

References 12 publications

Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene

Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene

Purification and Properties of d(−)‐3‐Hydroxybutyrate‐Dimer Hydrolase from Zoogloea ramigera I‐16‐M

Partial purification and characterization of ?-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium, Pseudomonas 135

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Purification and Properties of a D(-)-β-Hydroxybutyric Dimer Hydrolase from Rhodospirillum rubrum^*